The cAMP-responsive enhancer (CRE), originally characterized in our lab, serves as the target for the transcription factor CREB. Although CRE sequences have been identified in hundreds of genes, the factors that determine the specificity of CREB binding are still unknown. We have obtained the first insights into this issue by solving the structure of the CREB : CRE complex by x-ray crystallography. These data have allowed us to refine our concept of the essential nature of CREB and the CRE. CREB is activated by phosphorylation at Ser 133, which allows the association of the co-activator CREB binding protein (CBP). Divergent CRE sequences appear to recruit CBP through distinct mechanisms. We have studied one such element, located in the human T cell leukemia virus (HTLV)-1 promoter, and have determined that CREB and CBP binding to this site involves a virally encoded accessory protein called Tax, which overcomes the requirement for CREB phosphorylation. Our specific aims include: Elucidating the mechanisms underlying the specificity of CREB dimerization and binding, defining the essential features of the CRE, and determining what features define membership in the CREB/CREM/ATF-1 gene family. The CREB: CRE complex contains a cavity that may be filled by a hydrated Mg2+ ion, which may explain our earlier observation that Mg2+ was required for CREB binding to DNA. We will quantitate the Mg2+ dependence of the CREB : CRE interaction using fluorescence polarization assays and test the efficacy of other divalent cations. The crystal structure also predicts that a novel Tyr - Glu interaction might contribute importantly to dimerization. We will test this hypothesis by mutational analysis, measuring the ability of the mutated CREB proteins to dimerize and bind to DNA by using fluorescence resonance energy transfer, crosslinking, and fluorescence anisotropy determinations. We will also determine how the virally encoded Tax protein allows CREB to recognize the atypical CREs in the HTLV-1 promoter by characterizing the structure of the Tax: CREB : HTLV-1 DNA complex. Finally, we will characterize the ability of the "CREB binding domain" of CBP to interact with other transcription factors activated or inhibited by Ser/Thr phosphorylation.